scholarly journals Coxiella burnetii Expresses a Functional Δ24 Sterol Reductase

2010 ◽  
Vol 192 (23) ◽  
pp. 6154-6159 ◽  
Author(s):  
Stacey D. Gilk ◽  
Paul A. Beare ◽  
Robert A. Heinzen
Keyword(s):  
Host Cell ◽  
Coxiella Burnetii ◽  
Fluorescent Protein ◽  
De Novo ◽  
Cholesterol Metabolism ◽  
Q Fever ◽  
Parasitophorous Vacuole ◽  
Cholesterol Biosynthesis ◽  
Brefeldin A ◽  
Ergosterol Biosynthesis

ABSTRACT Coxiella burnetii, the etiological agent of human Q fever, occupies a unique niche inside the host cell, where it replicates in a modified acidic phagolysosome or parasitophorous vacuole (PV). The PV membrane is cholesterol-rich, and inhibition of host cholesterol metabolism negatively impacts PV biogenesis and pathogen replication. The precise source(s) of PV membrane cholesterol is unknown, as is whether the bacterium actively diverts and/or modifies host cell cholesterol or sterol precursors. C. burnetii lacks enzymes for de novo cholesterol biosynthesis; however, the organism encodes a eukaryote-like Δ24 sterol reductase homolog, CBU1206. Absent in other prokaryotes, this enzyme is predicted to reduce sterol double bonds at carbon 24 in the final step of cholesterol or ergosterol biosynthesis. In the present study, we examined the functional activity of CBU1206. Amino acid alignments revealed the greatest sequence identity (51.7%) with a Δ24 sterol reductase from the soil amoeba Naegleria gruberi. CBU1206 activity was examined by expressing the protein in a Saccharomyces cerevisiae erg4 mutant under the control of a galactose-inducible promoter. Erg4 is a yeast Δ24 sterol reductase responsible for the final reduction step in ergosterol synthesis. Like Erg4-green fluorescent protein (GFP), a CBU1206-GFP fusion protein localized to the yeast endoplasmic reticulum. Heterologous expression of CBU1206 rescued S. cerevisiae erg4 sensitivity to growth in the presence of brefeldin A and cycloheximide and resulted in new synthesis of ergosterol. These data indicate CBU1206 is an active sterol reductase and suggest the enzyme may act on host sterols during C. burnetii intracellular growth.

scholarly journals Sustained Axenic Metabolic Activity by the Obligate Intracellular Bacterium Coxiella burnetii

2008 ◽  
Vol 190 (9) ◽  
pp. 3203-3212 ◽  
Author(s):  
Anders Omsland ◽  
Diane C. Cockrell ◽  
Elizabeth R. Fischer ◽  
Robert A. Heinzen
Keyword(s):  
Host Cell ◽  
Coxiella Burnetii ◽  
Metabolic Activity ◽  
De Novo ◽  
Q Fever ◽  
Parasitophorous Vacuole ◽  
Chloride Concentration ◽  
Citrate Buffer ◽  
Cell Variant

ABSTRACT Growth of Coxiella burnetii, the agent of Q fever, is strictly limited to colonization of a viable eukaryotic host cell. Following infection, the pathogen replicates exclusively in an acidified (pH 4.5 to 5) phagolysosome-like parasitophorous vacuole. Axenic (host cell free) buffers have been described that activate C. burnetii metabolism in vitro, but metabolism is short-lived, with bacterial protein synthesis halting after a few hours. Here, we describe a complex axenic medium that supports sustained (>24 h) C. burnetii metabolic activity. As an initial step in medium development, several biological buffers (pH 4.5) were screened for C. burnetii metabolic permissiveness. Based on [35S]Cys-Met incorporation, C. burnetii displayed optimal metabolic activity in citrate buffer. To compensate for C. burnetii auxotrophies and other potential metabolic deficiencies, we developed a citrate buffer-based medium termed complex Coxiella medium (CCM) that contains a mixture of three complex nutrient sources (neopeptone, fetal bovine serum, and RPMI cell culture medium). Optimal C. burnetii metabolism occurred in CCM with a high chloride concentration (140 mM) while the concentrations of sodium and potassium had little effect on metabolism. CCM supported prolonged de novo protein and ATP synthesis by C. burnetii (>24 h). Moreover, C. burnetii morphological differentiation was induced in CCM as determined by the transition from small-cell variant to large-cell variant. The sustained in vitro metabolic activity of C. burnetii in CCM provides an important tool to investigate the physiology of this organism including developmental transitions and responses to antimicrobial factors associated with the host cell.

scholarly journals Coxiella burnetii Alters Cyclic AMP-Dependent Protein Kinase Signaling during Growth in Macrophages

2012 ◽  
Vol 80 (6) ◽  
pp. 1980-1986 ◽  
Author(s):  
Laura J. MacDonald ◽  
Richard C. Kurten ◽  
Daniel E. Voth
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Coxiella Burnetii ◽  
Alveolar Macrophages ◽  
Q Fever ◽  
Parasitophorous Vacuole ◽  
Dependent Protein Kinase ◽  
Content Type ◽  
Bacterial Agent ◽  
Dependent Protein

ABSTRACTCoxiella burnetiiis the bacterial agent of human Q fever, an acute, flu-like illness that can present as chronic endocarditis in immunocompromised individuals. Following aerosol-mediated transmission,C. burnetiireplicates in alveolar macrophages in a unique phagolysosome-like parasitophorous vacuole (PV) required for survival. The mechanisms ofC. burnetiiintracellular survival are poorly defined and a recent Q fever outbreak in the Netherlands emphasizes the need for better understanding this unique host-pathogen interaction. We recently demonstrated that inhibition of host cyclic AMP-dependent protein kinase (PKA) activity negatively impacts PV formation. In the current study, we confirmed PKA involvement in PV biogenesis and probed the role of PKA signaling duringC. burnetiiinfection of macrophages. Using PKA-specific inhibitors, we found the kinase was needed for biogenesis of prototypical PV andC. burnetiireplication. PKA and downstream targets were differentially phosphorylated throughout infection, suggesting prolonged regulation of the pathway. Importantly, the pathogen actively triggered PKA activation, which was also required for PV formation by virulentC. burnetiiisolates during infection of primary human alveolar macrophages. A subset of PKA-specific substrates were differentially phosphorylated duringC. burnetiiinfection, suggesting the pathogen uses PKA signaling to control distinct host cell responses. Collectively, the current results suggest a versatile role for PKA inC. burnetiiinfection and indicate virulent organisms usurp host kinase cascades for efficient intracellular growth.

scholarly journals Identification of ElpA, a Coxiella burnetii Pathotype-Specific Dot/Icm Type IV Secretion System Substrate

2015 ◽  
Vol 83 (3) ◽  
pp. 1190-1198 ◽  
Author(s):  
Joseph G. Graham ◽  
Caylin G. Winchell ◽  
Uma M. Sharma ◽  
Daniel E. Voth
Keyword(s):  
Coxiella Burnetii ◽  
Q Fever ◽  
Parasitophorous Vacuole ◽  
Secretion System ◽  
Terminal Region ◽  
Effector Proteins ◽  
Type Iv Secretion ◽  
Type Iv Secretion System ◽  
Content Type ◽  
Type Iv

Coxiella burnetiicauses human Q fever, a zoonotic disease that presents with acute flu-like symptoms and can result in chronic life-threatening endocarditis. In human alveolar macrophages,C. burnetiiuses a Dot/Icm type IV secretion system (T4SS) to generate a phagolysosome-like parasitophorous vacuole (PV) in which to replicate. The T4SS translocates effector proteins, or substrates, into the host cytosol, where they mediate critical cellular events, including interaction with autophagosomes, PV formation, and prevention of apoptosis. Over 100C. burnetiiDot/Icm substrates have been identified, but the function of most remains undefined. Here, we identified a novel Dot/Icm substrate-encoding open reading frame (CbuD1884) present in allC. burnetiiisolates except the Nine Mile reference isolate, where the gene is disrupted by a frameshift mutation, resulting in a pseudogene. The CbuD1884 protein contains two transmembrane helices (TMHs) and a coiled-coil domain predicted to mediate protein-protein interactions. The C-terminal region of the protein contains a predicted Dot/Icm translocation signal and was secreted by the T4SS, while the N-terminal portion of the protein was not secreted. When ectopically expressed in eukaryotic cells, the TMH-containing N-terminal region of the CbuD1884 protein trafficked to the endoplasmic reticulum (ER), with the C terminus dispersed nonspecifically in the host cytoplasm. This new Dot/Icm substrate is now termed ElpA (ER-localizingproteinA). Full-length ElpA triggered substantial disruption of ER structure and host cell secretory transport. These results suggest that ElpA is a pathotype-specific T4SS effector that influences ER function duringC. burnetiiinfection.

scholarly journals The Gamma Interferon (IFN-γ)-Inducible GTP-Binding Protein IGTP Is Necessary for Toxoplasma Vacuolar Disruption and Induces Parasite Egression in IFN-γ-Stimulated Astrocytes

2008 ◽  
Vol 76 (11) ◽  
pp. 4883-4894 ◽  
Author(s):  
T. Melzer ◽  
A. Duffy ◽  
L. M. Weiss ◽  
S. K. Halonen
Keyword(s):  
Host Cell ◽  
Fluorescent Protein ◽  
Binding Protein ◽  
Parasitophorous Vacuole ◽  
Central Nervous System Infection ◽  
Gamma Interferon ◽  
Gtp Binding Protein ◽  
Gtp Binding ◽  
Ifn Γ ◽  
Green Fluorescent

ABSTRACT Toxoplasma gondii is a common central nervous system infection in individuals with immunocompromised immune systems, such as AIDS patients. Gamma interferon (IFN-γ) is the main cytokine mediating protection against T. gondii. Our previous studies found that IFN-γ significantly inhibits T. gondii in astrocytes via an IFN-γ-inducible GTP-binding protein (IGTP)-dependent mechanism. The IGTP-dependent-, IFN-γ-stimulated inhibition is not understood, but recent studies found that IGTP induces disruption of the parasitophorous vacuole (PV) in macrophages. In the current study, we have further investigated the mechanism of IFN-γ inhibition and the role of IGTP in the vacuolar disruption in murine astrocytes. Vacuolar disruption was found to be dependent upon IGTP, as PV disruption was not observed in IGTP-deficient (IGTP−/−) astrocytes and PV disruption could be induced in IGTP−/− astrocytes transfected with IGTP. Live-cell imaging studies using green fluorescent protein-IGTP found that IGTP is delivered to the PV via the host cell endoplasmic reticulum (ER) early after invasion and that IGTP condenses into vesicle-like structures on the vacuole just prior to PV disruption, suggesting that IGTP is involved in PV disruption. Intravacuolar movement of the parasite occurred just prior to PV disruption. In some instances, IFN-γ induced parasite egression. Electron microscopy and immunofluorescence studies indicate that the host cell ER fuses with the PV prior to vacuolar disruption. On the basis of these results, we postulate a mechanism by which ER/PV fusion is a crucial event in PV disruption. Fusion of the ER with the PV, releasing calcium into the vacuole, may also be the mechanism by which intravacuolar parasite movement and IFN-γ-induced parasite egression occur.

scholarly journals Sustained Activation of Akt and Erk1/2 Is Required for Coxiella burnetii Antiapoptotic Activity

2008 ◽  
Vol 77 (1) ◽  
pp. 205-213 ◽  
Author(s):  
Daniel E. Voth ◽  
Robert A. Heinzen
Keyword(s):  
Host Cell ◽  
Coxiella Burnetii ◽  
Stress Responses ◽  
Mammalian Cells ◽  
Apoptotic Cell ◽  
Parasitophorous Vacuole ◽  
Critical Role ◽  
Bacterial Pathogen ◽  
Human Macrophage ◽  
Antiapoptotic Activity

ABSTRACT Coxiella burnetii is an obligate intracellular bacterial pathogen that directs biogenesis of a lysosome-like, parasitophorous vacuole in mammalian cells. We recently reported that C. burnetii inhibits apoptotic cell death in macrophages, presumably as a mechanism to sustain the host for completion of its lengthy infectious cycle. In the current study, we further investigated C. burnetii manipulation of host cell signaling and apoptosis by examining the effect of C. burnetii infection on activation of 15 host proteins involved in stress responses, cytokine production, and apoptosis. C. burnetii infection of THP-1 human macrophage-like cells caused increased levels of phosphorylated c-Jun, Hsp27, Jun N-terminal protein kinase, and p38 at 2 h postinfection (hpi), and this activation rapidly decreased to near basal levels by 24 hpi. The prosurvival kinases Akt and Erk1/2 (extracellular signal-regulated kinases 1 and 2) were also activated at 2 to 6 hpi; however, the phosphorylation of these proteins increased coincident with C. burnetii replication through at least 72 hpi. Sustained phosphorylation of Akt and Erk1/2 was abolished by treatment of infected cells with rifampin, indicating their activation is a C. burnetii-directed event requiring pathogen RNA synthesis. Moreover, pharmacological inhibition of Akt or Erk1/2 significantly decreased C. burnetii antiapoptotic activity. Collectively, these results indicate the importance of C. burnetii modulation of host signaling and demonstrate a critical role for Akt and Erk1/2 in successful intracellular parasitism and maintenance of host cell viability.

scholarly journals Dependency of Coxiella burnetii Type 4B Secretion on the Chaperone IcmS

2019 ◽  
Vol 201 (23) ◽  
Author(s):  
Charles L. Larson ◽  
Paul A. Beare ◽  
Robert A. Heinzen
Keyword(s):  
Host Cell ◽  
Coxiella Burnetii ◽  
Legionella Pneumophila ◽  
Q Fever ◽  
Secretion System ◽  
Host Cells ◽  
Growth Defect ◽  
Wild Type ◽  
Signal Sequences ◽  
Content Type

ABSTRACT Macrophage parasitism by Coxiella burnetii, the cause of human Q fever, requires the translocation of proteins with effector functions directly into the host cell cytosol via a Dot/Icm type 4B secretion system (T4BSS). Secretion by the analogous Legionella pneumophila T4BSS involves signal sequences within the C-terminal and internal domains of effector proteins. The cytoplasmic chaperone pair IcmSW promotes secretion and binds internal sites distinct from signal sequences. In the present study, we investigated requirements of C. burnetii IcmS for host cell parasitism and effector translocation. A C. burnetii icmS deletion mutant (ΔicmS) exhibited impaired replication in Vero epithelial cells, deficient formation of the Coxiella-containing vacuole, and aberrant T4BSS secretion. Three secretion phenotypes were identified from a screen of 50 Dot/Icm substrates: IcmS dependent (secreted by only wild-type bacteria), IcmS independent (secreted by both wild-type and ΔicmS bacteria), or IcmS inhibited (secreted by only ΔicmS bacteria). Secretion was assessed for N-terminal or C-terminal truncated forms of CBU0794 and CBU1525. IcmS-inhibited secretion of CBU1525 required a C-terminal secretion signal whereas IcmS-dependent secretion of CBU0794 was directed by C-terminal and internal signals. Interchange of the C-terminal 50 amino acids of CBU0794 and CBU1525 revealed that sites within the C terminus regulate IcmS dependency. Glutathione S-transferase-tagged IcmSW bound internal sequences of IcmS-dependent and -inhibited substrates. Thus, the growth defect of the C. burnetii ΔicmS strain is associated with a loss of T4BSS chaperone activity that both positively and negatively regulates effector translocation. IMPORTANCE The intracellular pathogen Coxiella burnetii employs a type 4B secretion system (T4BSS) that promotes growth by translocating effectors of eukaryotic pathways into host cells. T4BSS regulation modeled in Legionella pneumophila indicates IcmS facilitates effector translocation. Here, we characterized type 4B secretion by a Coxiella ΔicmS mutant that exhibits intracellular growth defects. T4BSS substrates demonstrated increased, equivalent, or decreased secretion by the ΔicmS mutant relative to wild-type Coxiella. Similar to the Legionella T4BSS, IcmS dependency in Coxiella was determined by C-terminal and/or internal secretion signals. However, IcmS inhibited secretion of some effectors by Coxiella that were previously shown to be translocated by Legionella. Thus, Coxiella has a unique IcmS regulatory mechanism that both positively and negatively regulates T4BSS export.

scholarly journals Development of anEx VivoTissue Platform To Study the Human Lung Response to Coxiella burnetii

2016 ◽  
Vol 84 (5) ◽  
pp. 1438-1445 ◽  
Author(s):  
Joseph G. Graham ◽  
Caylin G. Winchell ◽  
Richard C. Kurten ◽  
Daniel E. Voth
Keyword(s):  
Coxiella Burnetii ◽  
Lung Tissue ◽  
Human Lung ◽  
Q Fever ◽  
Ex Vivo ◽  
Parasitophorous Vacuole ◽  
Inflammasome Activation ◽  
Human Lung Tissue ◽  
Content Type

Coxiella burnetiiis an intracellular bacterial pathogen that causes human Q fever, an acute debilitating flu-like illness that can also present as chronic endocarditis. Disease typically occurs following inhalation of contaminated aerosols, resulting in an initial pulmonary infection. In human cells,C. burnetiigenerates a replication niche termed the parasitophorous vacuole (PV) by directing fusion with autophagosomes and lysosomes.C. burnetiirequires this lysosomal environment for replication and uses a Dot/Icm type IV secretion system to generate the large PV. However, we do not understand howC. burnetiievades the intracellular immune surveillance that triggers an inflammatory response. We recently characterized human alveolar macrophage (hAM) infectionin vitroand found that avirulentC. burnetiitriggers sustained interleukin-1β (IL-1β) production. Here, we evaluated infection ofex vivohuman lung tissue, defining a valuable approach for characterizingC. burnetiiinteractions with a human host. Within whole lung tissue,C. burnetiipreferentially replicated in hAMs. Additionally, IL-1β production correlated with formation of an apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (ASC)-dependent inflammasome in response to infection. We also assessed potential activation of a human-specific noncanonical inflammasome and found that caspase-4 and caspase-5 are processed during infection. Interestingly, although inflammasome activation is closely linked to pyroptosis, lytic cell death did not occur followingC. burnetii-triggered inflammasome activation, indicating an atypical response after intracellular detection. Together, these studies provide a novel platform for studying the human innate immune response toC. burnetii.

scholarly journals The Coxiella burnetii Ankyrin Repeat Domain-Containing Protein Family Is Heterogeneous, with C-Terminal Truncations That Influence Dot/Icm-Mediated Secretion

2009 ◽  
Vol 191 (13) ◽  
pp. 4232-4242 ◽  
Author(s):  
Daniel E. Voth ◽  
Dale Howe ◽  
Paul A. Beare ◽  
Joseph P. Vogel ◽  
Nathan Unsworth ◽  
...  
Keyword(s):  
Genome Sequencing ◽  
Coxiella Burnetii ◽  
Legionella Pneumophila ◽  
Mammalian Cells ◽  
Q Fever ◽  
Parasitophorous Vacuole ◽  
Protein Family ◽  
Ankyrin Repeat ◽  
Reference Isolate ◽  
Type Iv

ABSTRACT Coxiella burnetii is an obligate intracellular bacterium that directs biogenesis of a parasitophorous vacuole (PV) for replication. Effectors of PV maturation are likely translocated into the host cytosol by a type IV secretion system (T4SS) with homology to the Dot/Icm apparatus of Legionella pneumophila. Since secreted bacterial virulence factors often functionally mimic the activities of host proteins, prokaryotic proteins with eukaryotic features are considered candidate T4SS substrates. Genes encoding proteins with eukaryotic-type ankyrin repeat domains (Anks) were identified upon genome sequencing of the C. burnetii Nine Mile reference isolate, which is associated with a case of human acute Q fever. Interestingly, recent genome sequencing of the G and K isolates, derived from human chronic endocarditis patients, and of the Dugway rodent isolate revealed remarkable heterogeneity in the Ank gene family, with the Dugway isolate harboring the largest number of full-length Ank genes. Using L. pneumophila as a surrogate host, we identified 10 Dugway Anks and 1 Ank specific to the G and K endocarditis isolates translocated into the host cytosol in a Dot/Icm-dependent fashion. A 10-amino-acid C-terminal region appeared to be necessary for translocation, with some Anks also requiring the chaperone IcmS for secretion. Ectopically expressed Anks localized to a variety of subcellular regions in mammalian cells, including microtubules, mitochondria, and the PV membrane. Collectively, these data suggest that C. burnetii isolates translocate distinct subsets of the Ank protein family into the host cytosol, where they modulate diverse functions, some of which may be unique to C. burnetii pathotypes.

scholarly journals The secreted protein kinase CstK from Coxiella burnetii influences vacuole development and interacts with the GTPase-activating host protein TBC1D5

2020 ◽  
Vol 295 (21) ◽  
pp. 7391-7403 ◽  
Author(s):  
Eric Martinez ◽  
Sylvaine Huc-Brandt ◽  
Solène Brelle ◽  
Julie Allombert ◽  
Franck Cantet ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Coxiella Burnetii ◽  
Membrane Trafficking ◽  
Q Fever ◽  
Parasitophorous Vacuole ◽  
Host Protein ◽  
Host Cells ◽  
Intracellular Replication ◽  
Gtpase Activating Protein ◽  
Infected Cells

The intracellular bacterial pathogen Coxiella burnetii is the etiological agent of the emerging zoonosis Q fever. Crucial to its pathogenesis is type 4b secretion system–mediated secretion of bacterial effectors into host cells that subvert host cell membrane trafficking, leading to the biogenesis of a parasitophorous vacuole for intracellular replication. The characterization of prokaryotic serine/threonine protein kinases in bacterial pathogens is emerging as an important strategy to better understand host–pathogen interactions. In this study, we investigated CstK (for Coxiella Ser/Thr kinase), a protein kinase identified in C. burnetii by in silico analysis. We demonstrate that this putative protein kinase undergoes autophosphorylation on Thr and Tyr residues and phosphorylates a classical eukaryotic protein kinase substrate in vitro. This dual Thr-Tyr kinase activity is also observed for a eukaryotic dual-specificity Tyr phosphorylation-regulated kinase class. We found that CstK is translocated during infections and localizes to Coxiella-containing vacuoles (CCVs). Moreover, a CstK-overexpressing C. burnetii strain displayed a severe CCV development phenotype, suggesting that CstK fine-tunes CCV biogenesis during the infection. Protein–protein interaction experiments identified the Rab7 GTPase-activating protein TBC1D5 as a candidate CstK-specific target, suggesting a role for this host GTPase-activating protein in Coxiella infections. Indeed, CstK co-localized with TBC1D5 in noninfected cells, and TBC1D5 was recruited to CCVs in infected cells. Accordingly, TBC1D5 depletion from infected cells significantly affected CCV development. Our results indicate that CstK functions as a bacterial effector protein that interacts with the host protein TBC1D5 during vacuole biogenesis and intracellular replication.

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